ABSTRACT Stroke is the fifth leading cause of death and the leading cause of disability in the United States. There remains a critical need for innovative therapeutic approaches that can successfully prevent or reverse brain injury following stroke. Dysfunction of the mitochondrial biochemistry following ischemic stroke and reperfusion injury contributes to significant neuronal cell loss; however, the development of therapeutics targeting mitochondrial function for stroke patients are lacking. Mitochondrial dysfunction plays a central role in the neuronal cell death seen in ischemia-reperfusion injury, but has not yet been fully investigated as drug target. In this project, we are investigating novel mitochondrial protein mitoNEET as therapeutic drug target of mitochondrial function in stroke. MitoNEET is a newly discovered protein that regulates mitochondrial bioenergetics. We developed a first-in- class mitoNEET agonist NL-1 which showed significant tissue protection after transient ischemia in the brain. The objectives of this proposal are to evaluate a recently discovered mitochondrial protein, mitoNEET, as an effective therapeutic approach for the pharmacological treatment of stroke. MitoNEET (CISD1 gene) regulates mitochondrial bioenergetics capacity where it functions as redox sensor. Our central hypothesize is that ligands selective for mitoNEET will protect brain tissue from hypoxia-induced reperfusion injury. Our approach involves state of the art medicinal chemistry approaches to identify novel drug candidates and develop a structure-activity relationship. In our first aim, we will develop potent and selective mitoNEET ligands with drug-like properties. In the second aim, we will optimize the formulation of the mitoNEET ligands for in vivo studies. These compounds and drug delivery systems will have far-reaching implication for developing neuroprotective medications as a treatment strategy for limiting the mitochondrial contribution to cell loss in the clinical setting in the treatment of stroke patients.